Smear Lab Report

The experimental part of the lab consists of setting up the materials needed. A sample of E.coli and a solution of calcium chloride are first obtained and placed in different test tubes. 630µL of Calcium Chloride (CaCl2) are then removed from the test tube and inserted into the test tube containing E.coli cells (Alberte et al., 2012). The newly formed substance of Calcium Chloride and E.coli is then mixed and incubated in ice for 10 minutes, making the cells more competent. Two test tubes are obtained and labeled; the first test tube is labeled with pUC18 and the second one with “Lux” to represent the plasmids being used. These two test tubes are then incubated in ice. 3µl of the set plasmid are added to each of the two test tubes. The test tubes are tapped to guarantee the cells are well

To perform this test, a small drop of water is placed on a clean microscope slide. A metal loop that has been properly sterilized in the blue flame and allowed time to cool is used to

1) Apply the stain to your first unknown slide and examine it under the microscope.

To perform this test, a tube of broth rich with glucose is acquired. In this tube is phenol red, a pH indicator. Initially, the tube appeared pink in color, indicating a normal pH level. Next, a sample of unknown #44 is introduced into this medium using the aseptic technique, and this is allowed to sit for several days. If the organism is able to ferment glucose, the pH in the medium would decrease and cause the phenol red to exhibit a yellow color. In addition to the straw color, gas can also be produced and trapped inside the Durham tube placed in the medium. This production of acid and gas is a direct result of the fermentation of glucose, as seen with unknown

5 Remaining in the darkroom move print to tray of running water, do 30-45 second rinse. Squeegee print, place on flat tray.

Lastly, safranin was added to flood the slide and was left on for 1 minute and rinsed with distilled water. After adding all 4 reagents, the slide was blotted dry with a paper towel to remove any excess reagents or water. The slide was then placed on a microscope stage to look for the color of the bacteria. When ready for the highest power on the microscope, immersion oil was added to the slide and looked at under 100x power. A glucose test and citrate test were found to be the best two tests to help narrow down the bacteria.

As soon as the water was added a timer was set for two minutes and the slide was placed under the microscope.

This is important in the medical field because identification of unknown bacteria can help treat a patient by knowing the contributing source of a disease. Also knowledge of different bacteria helped others make antibiotics used today. This lab was completed by using the methods learned thus far in identification of bacteria.

There were two tubes used in this process: the tube that contained the primary culture and the tube that contained the nutrient agar where the unknown bacteria would grow. First, the inoculating loop was flamed. After removing the caps of both the test tubes, they were flamed to prevent contamination of the unknown bacteria. The inoculating loop was cooled for a few seconds and was then placed into the test tube containing the bacteria. The inoculating loop with the bacteria was placed into the nutrient agar test tube for cultivation. Before the test tubes were capped, they were flamed once again. Also, isolation of the unknown bacteria had to completed. Nutrient agar was placed in the petri dish, and was left to gel for a few minutes. After the agar gelled, the inoculating loop was used to acquire bacteria and streak the unknown onto the plate for

6. The disks in the 0.00% solution were transferred to an agar plate held next to the blue flame using the sterilized tweezers. Excess disinfectant was removed from the disks by wiping on the side of the well of the spotting tile. When the 5 disks were positioned (refer to Figure 1 below) the lid was replaced and sticky taped down. A label was added indicating the concentration of disinfectant.

And the label with a positive sign (+) before pGLO indicates the presence of the plasmid pGLO. Next I put on a pair of vinyl gloves, which would stay on my hands for the entirety of the experiment for safety. Next using a micropipetter we transferred 250 µL of LB nutrient broth into each tube, using a fresh tip for each transfer. The lids of the labeled tubes were then closed and the tubes were put into an ice bath. While the tubes were cooling we used a sterile loop to brush against bacteria E. Coli starter plate picking up a single colony of bacteria.

Figure #1 This picture show the gram stain of unknown #3. The image is under 1000x magnification using oil immersion technique. Gram (-)

The drops of crystal violets, approximately 15 drops, were flooded until the smear were all covered and then allowing resting for one to two minutes. After two minutes, the slide was titled over the sink and washed off, with the distilled water bottle, by aiming the stream of water above the smear. The specimen appeared blue-violet when observed with the naked eye. The drops of Gram’s iodine were applied on the slide until covered and then allowed to react for one minute or more. After the time elapsed, the slide was rinsed again with distilled water following immediate drops of Gram stain decolorizer added one drop at a time.

Turn the starch agar plates upside down and draw two lines with the sharpie on each, dividing each petri dish into four separate sections. Label the sections 1A, 1B, 1C, and 1D on the first petri dish, 2A, 2B, 2C, and 2D on the second, and so on. 2. Pass forceps through the Bunsen burner flame; let them cool for a little bit, then use them to pick up one of the paper discs. Open the culture of Bacillus subtilis, flame the neck of the bottle, and dip the disc in the broth.

The petri dish lid was raised to insert the loop. The loop was touched to the agar area on the opposite side of the dish. The bacteria on the loop were transferred to the agar. The bacteria were spread in the first sector of the petri dish by moving the loop back and forth across the dish (zigzag motion).